Identification and functional characterization of novel thioredoxin systems

Thioredoxins (Trx) are a class of small multifunctional 12-kDa proteins that are characterized by the redox active site Trp-Cys-Gly-Pro-Cys (WCGPC). In the oxidized (inactive) form of Trx (Trx-S2), the two cysteines at the active site form a disulfide bond. This can then be reduced by thioredoxin reductase (TrxR) and NADPH, the so-called thioredoxin system, to a dithiol (Trx-(SH)2), which can then act as a general protein disulfide reductase. Thioredoxins are present in all living organisms and have been isolated and characterized from a wide variety of prokaryotic and eukaryotic cells. In this thesis we describe the identification and functional characterization of novel members of the thioredoxin superfamily.

We present evidence for a novel Trx (Trx2) in Escherichia coli. The E. coli Trx2 contains two domains: an N-terminal domain of 32 amino acids containing two CXXC motifs and a C-terminal domain with high homology to the prokaryotic thioredoxins, containing the conserved active site. Trx2 together with TrxR and NADPH can reduce ribonucleotide reductase as well as the interchain disulfide bridges of insulin.

Thioredoxins are ubiquitously expressed in an tissues within the same organism. We have identified the first tissue specific Trx (Sptrx1) exclusively expressed in human spermatozoa. Sptrx1 is an active thioredoxin which under native conditions appears to have a multimeric structure. We also identify and characterize a complete thioredoxin system (Trx2, TrxR2) located in mitochondria. We show that Trx2 overexpressing cells have a higher mitochondrial membrane potential that is dependent on the function of the ATP synthase complex. Furthermore, overexpression of Trx2 was found to protect cells against the cytotoxic effects of etoposide, a drug commonly used in anticancer treatment.

In addition, we showed that the second compound of the mitochondrial thioredoxin system, TrxR2, is capable of reducing cytochrome c and could protect cells against the cytotoxic effects of antimycin and myxothiazol, chemicals that inhibit the function of complex III in the mitochondrial electron transport chain. Furthemore, we identified an alternative splicing variant of cytosolic thioredoxin reductase (TrxR1b) that could bind to the Estrogen Receptors (ER) alpha and beta. As a result of this binding, a distinct subnuclear localization of TrxR1b was observed, co localizing with both ER alpha and beta. TrxR1b can act as a coactivator and enhance the transcriptional activity of ER in the classical activation pathway, which relies on the binding of the ER to an ER response element on the DNA. By contrast, TrxR1b is a co-repressor in the alternative pathway where ER activates AP-I transcription independently of its DNA binding activity.

In summary, the results presented in this thesis give a better understanding of Thioredoxin systems in both prokaryotes and eukaryotes, with the introduction of new members in this redox superfamily of proteins. This study, which shows a wide spectrum of functions for these Thioredoxin systems in influencing various redox mechanisms and processess in biological systems, indicates that there is still a great deal of work yet to be done in this expanding field of research.

List of scientific papers

I. Miranda-Vizuete A, Damdimopoulos AE, Gustafsson J, Spyrou G (1997). Cloning, expression, and characterization of a novel Escherichia coli thioredoxin. J Biol Chem. 272(49): 30841-7.
https://pubmed.ncbi.nlm.nih.gov/9388228

II. Miranda-Vizuete A, Ljung J, Damdimopoulos AE, Gustafsson JA, Oko R, Pelto-Huikko M, Spyrou G (2001). Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa. J Biol Chem. 276(34): 31567-74. Epub 2001 Jun 08
https://pubmed.ncbi.nlm.nih.gov/11399755

III. Damdimopoulos AE, Miranda-Vizuete A, Pelto-Huikko M, Gustafsson JA, Spyrou G (2002). Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death. J Biol Chem. 277(36): 33249-57. Epub 2002 Jun 21
https://pubmed.ncbi.nlm.nih.gov/12080052

IV. Miranda-Vizuete A, Damdimopoulos AE, Pedrajas JR, Gustafsson JA, Spyrou G (1999). Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur J Biochem. 261(2): 405-12.
https://pubmed.ncbi.nlm.nih.gov/10215850

V. Nalvarte I, Damdimopoulos AE, Spyrou G (2003). Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition. [Manuscript]

VI. Damdimopoulos AE, Miranda-Vizuete A, Treuter E, Gustafsson JA, Spyrou G (2003). The selenoprotein thioredoxin reductase as a modulator of estrgen signaling. [Manuscript]